Nicotinic receptors. Nicotinic acetylcholine receptors (nAChRs) are composed of various combinations of α-subunits (α2–α9) and β-subunits (β2–β4), and are classified into two classes according to their affinity for nicotine or α-bungarotoxin (ABTX) (Vijayaraghavan, S., et al., Neuron 8:353–362 (1992)). Of the known αBTX-binding subtypes α7–α9, only α7 receptors are expressed throughout the mammalian brain. Alpha7 receptors form functional homomeric ion channels that promote Ca2+ influx, which are rapidly desensitized (Breese, C. R., et al., J. Com. Neurol. 387:385–398 (1997); Vijayaraghavan, S., et al., Neuron 8:353–362 (1992)) and are thus assumed to be involved in synaptic transmission (McGehee, D. S., et al., Science 269:1692–1696 (1995)). Nicotinic agonists selective for the α7 receptor have demonstrated efficacy in improving cognitive functions in rats, primates and AD patients. Due to the multifaceted deficits observed in AD and the limited pharmacopae for management of AD patients, there is an urgent need for new therapies and approaches to optimize existing and emerging therapies.
Nicotine has also been found to inhibit death of PC12 cells cultured in serum-free medium (Yamashita, H. and S. Nakamura, Neurosci. Lett. 213:145–147 (1996)). In addition, a selective α7 receptor agonist, anabaseine-derived 3-(4)-dimethylaminocinamylidine (DMAC) (de Fiebre, C. M., et al., Mol. Pharmacol. 47:164–171 (1995)), and an activator of nAChR, including the α7 subtype, ABT-418 (Donnelly-Roberts, D. L., et al., Brain Res. 719:36–44 (1996)) have also been reported to exert cytoprotective effects.
Nicotine-induced protection in neuronal cells is suppressed by αBTX, a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002 and wortmannin), and a Src inhibitor (PP2). In addition, the levels of phosphorylated Akt, an effector of PI3K, are increased by nicotine ((Kihara, T., S. et al., J. Biol. Chem. 276:13541–13546 (2001)). These findings suggest that the α7 nicotinic receptor transduces signals to PI3K in a cascade, which ultimately contributes to a neuroprotective effect.
Ang II signaling pathways. The actions of Angiotensin II (Ang II) are mediated through two types of cell surface receptors, (AT1 and AT2). Most of the physiological responses to Ang II in glomerular mesangial cells (GMC) occur via the AR1 receptor subtype (Bernstein, K. E. and M. B. Marrero, Trends. Cardiovasc. Med. 6:179–187 (1996); Marrero, M. B., et al., Cell. Signal. 8:21–26 (1996)). For AR1 receptors, activation by Ang II results in G protein mediated signaling, including phospholipase C-dependent activation of protein kinase C and release of calcium from intracellular stores (Bernstein, K. E. and M. B. Marrero, Trends. Cardiovasc. Med. 6:179–187 (1996)). AT1 receptors also activate signaling pathways traditionally associated with growth factor and cytokine receptors that induce the production of early growth response genes. The signaling cascades whereby Ang II induces early growth response genes, such as c-fos and c-jun proto-oncogenes, does not in general require new protein synthesis and appear to be regulated by post-translational modifications of pre-existing transcription factors (Sadoshima, J. and S. Izumo, Circ. Res. 73:413–423 (1993); Okuda, M., Y. Kawahara, and M. Yokoyama, Am. J. Physiol. 271: H595–H601, (1996); Sadoshima, J. and S. Izumo, Circ. Res. 73:413–423 (1993); Sadoshima, J. and S. Izumo, Circ. Res. 73:424–438 (1993); Taubman, M. B., et al., J. Biol. Chem. 264:526–530 (1989)). Therefore, the Ang II-induced expression of these early growth response genes is under the direct regulation of intracellular signal transduction pathways. Three intracellular signaling pathways have recently been implicated in the activation of proto-oncogenes: the JAK/STAT, p21ras/Raf-1/MAP kinase, and the PLC-γ1 cascades (Bernstein, K. E. and M. B. Marrero, Trends. Cardiovasc. Med. 6:179–187 (1996); Marrero, M. B., et al., Cell. Signal. 8:21–26 (1996); Sayeski, P. P., et al. Regulatory Peptides 78:19–29 (1998)). From multiple studies focusing on AR1 receptor signal transduction pathways, it has become apparent that the temporal arrangement of agonist-stimulated signaling varies from seconds (i.e., the activation of PLC-γ1 and generation of inositol phosphates) to minutes (e.g., MAP kinase activation) to hours (e.g., JAK/STAT pathway) (Bernstein, K. E. and M. B. Marrero, Trends. Cardiovasc. Med. 6:179–187 (1996); Sayeski, P. P., et al., Regulatory Peptides 78:19–29 (1998)). The exact mechanism(s) by which the AT1 receptor is able to differentially couple to disparate signal transduction pathways is not clear, but presumably involves a complex series of steps that selectively recruits, activates and then inactivates each signaling system in a time-dependent manner.
Role of the JAK/STAT Pathway in Ang II Signaling. The JAK family of cytosolic tyrosine kinases, traditionally thought to be coupled to cytokine receptors such as those for the interleukins and interferons, have four members (JAK1, JAK2, JAK3 and TYK2) (Darnell, J. E., Jr., et al., Science 264:1415–1421 (1994); Taubman, M. B., et al., J. Biol. Chem. 264:526–530 (1989)). In response to ligand binding, these JAK tyrosine kinases associate with, tyrosine-phosphorylate, and activate the cytokine receptor itself. Once activated, JAKs tyrosine-phosphorylate and activate other signaling molecules including the STAT family of nuclear transcription factors after binding of the STATs to the receptor (Darnell, J. E., Jr., et al., Science 264:1415–1421 (1994); Taubman, M. B., et al., J. Biol. Chem. 264:526–530 (1989)). Thus, the JAK/STAT pathway is an important link between cell surface receptors and nuclear transcriptional events leading to cell growth. Recently, Baker and colleagues have shown that STAT1, STAT3, and STAT5 are tyrosine-phosphorylated in response to Ang II in cardiac fibroblasts and AT1 receptor-transfected CHO cells (Bhat, G. J., et al., J. Biol. Chem. 269:31443–31449 (1994); Bhat, G. J., et al., J. Biol. Chem. 270:19059–19065 (1995); McWhinney, C. D., et al., J. Mol. Cell. Cardiol. 30:751–761 (1998)). These investigators also found that Ang II exposure stimulated the phosphorylated monomeric STAT proteins to form homo-(STAT12, STAT32 or STAT52) or hetero-(STAT1:STAT3) dimer complexes referred to as SIF (sis-inducing factors). These SIF complexes subsequently translocate to the nucleus and interact with specific DNA motifs called SIE (sis-inducing elements) or PIE (prolactin-inducing element)-like elements within the c-fos promoter, culminating in the activation of this early growth response gene (Bhat, G. J., et al., J. Biol. Chem. 269:31443–31449 (1994); Darnell, J. E., Jr., et al., Science 264:1415–1421 (1994); McWhinney, C. D., et al., J. Mol. Cell. Cardiol. 30:751–761 (1998); Schindler, C. and J. E. Darnell, Jr., Annu. Rev. Biochem. 64:621–651 (1995)). The JAK/STAT cascade can be activated by Ang II resulting in tyrosine phosphorylation of JAK2, STAT1 and STAT3, and the translocation of STAT1 and STAT3 to the nucleus (Bhat, G. J., et al., J. Biol. Chem. 270:19059–19065 (1995); Marrero, M. B., et al. Clin. Exp. Pharmacol. Physiol. 23:83–88 1996; Marrero, M. B., et al., Nature 375:247–250 (1995)). Furthermore, the carboxyl-terminal tail of the AT1 receptor binds to JAK2 in an Ang II-dependent manner (Ali, M. S., et al., J. Biol. Chem. 272:23382–23388 (1997)). In addition, inhibition of JAK2 tyrosine phosphorylation with the pharmacologic JAK2 inhibitor, AG490, or electroporation of blocking antibodies against STAT1 or STAT3 inhibits Ang II-induced vascular smooth muscle cell (VSMC) proliferation and DNA synthesis (Marrero, M. B., et al., J. Biol. Chem. 272:24684–24690 (1997)). These results indicate that G-protein-coupled receptors, in particular the AT1 receptor, can operate via the same intracellular tyrosine phosphorylation pathways previously linked to mitogenic cytokine and growth factor receptors. Finally, the tyrosine phosphatases, SHP-1 and SHP-2, have opposite roles in Ang II-induced JAK2 phosphorylation. SHP-1 appears responsible for JAK2 dephosphorylation and termination of the Ang II-induced JAK/STAT cascade, whereas SHP-2 appears to have an essential role in JAK2 phosphorylation and initiation of the Ang II-induced JAK/STAT cascade leading to cell proliferation (See Jiao, H., et al., Direct association with and dephosphorylation of Jak2 kinase by the SH2-domain-containing protein tyrosine phosphatase SHP-1. Mol Cell Biol 16(12):6985–92(1996)).
The motif in the AR1 receptor that is required for association with JAK2 is also required for association with SHP-2 (Marrero, M. B., et al., Am. J. Physiol. 275:C1216–C1223 (1998)). Furthermore, SHP-2 is also required for JAK2-Ang II AR1 receptor association (Marrero, M. B., et al., Am. J. Physiol. 275:C1216–C1223 (1998)). SHP-2 may thus play a role as an adaptor protein for JAK2 association with the receptor, thereby facilitating JAK2 phosphorylation and activation (Marrero, M. B., et al., Am. J. Physiol. 275: C1216–C1223, 1998).
Nicotinic Acetylcholine Receptors and β-Amyloid Toxicity. The cholinergic deficit in Alzheimer's Disease has been clearly established and is the basis for the current symptomatic strategy. There is an early and significant depletion of high affinity nicotinic receptors in Alzheimer's patient's brains (Court, J., et al. Biol. Psychiatry 49:175–184 (2001)), and a number of studies have shown cognitive improvement in rodent, primates including humans following administration of ligands targeting nAChRs (Newhouse Pa., et al., Biol Psychiatry 49(3):268–78 (2001)). In addition to their known symptomatic effects, neuronal nicotinic ligands have shown neuroprotective activity in vitro (Donnelly-Roberts, D. L., et al. Brain Res. 719: 36–44 (1996)) and in vivo (Ryan RE, et al., 132(8):1650–6 (2001)) suggesting an additional potential for disease modification.
The α7 receptor forms functional homomeric ligand-gated ion channels that promote rapidly desensitizing Ca2+ influx, is widely expressed throughout the mammalian brain, and has been implicated in sensory gating, cognition, and neuroprotection (Sco, J., et al., Biol Psychiatry 49(3):240–7 (2001). Nicotine-induced neuroprotection against β-Amyloid-induced toxicity is suppressed by α-Bgt and a selective α7-nAChR agonist, anabaseine-derived 3-(4)-dimethylaminocinamylidine (DMAC) exerts cytoprotective effects (De Fiebre C. M., et al., Mol. Pharmacol. 47:164–171 (1995); Kem W R., Behav Brain Res. 113(1–2):169–81 (2000)). The level of phosphorylated Akt, an effector of PI-3-K, is increased by nicotine and cytoprotective effects are suppressed by phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 and wortmannin), and Src inhibitor (PP2) (Kihara, T., et al., J. Biol. Chem. 276:13541–13546 (2001)). The α7-nAChR transduces signals to PI3K in a cascade, which ultimately contributes to a neuroprotective effect against Aβ.
In contrast to the decrease in α7-nAChR, the angiotensin converting enzyme (ACE—the enzyme that converts Angiotensin I to Angiotensin II) density is increased in the temporal cortex from patients with Alzheimer's disease (Barnes, N. M., et al., Eur. J. Pharmacol. 200:289–292 (1991)), and the ACE genotype is associated with AD in some populations (Narain Y et al., J Med Genet., 37(9):695–7 (2000)). AT2 receptors exert growth inhibitory effects or apoptosis both in cultured cells and in vivo (Horiuchi, M., et al., Endocr. Res. 24:307–314 (1998)), are expressed in PC12 cells, and have been shown to inhibit the JAK/STAT signaling cascade (Horiuchi, M., et al., Circ. Res. 84:876–882 (1999)).
It would be desirable to further understand any relationships between α7 nAChR-mediated beneficial pathways and the apoptotic effects mediated by AT2, in order to maximize cell survival by modulation of nAChR and/or AT2 activity.
It would also be desirable to further understand any relationship between Aβ-mediated toxicity and signaling pathways affected by nicotinic receptors. For example, further elucidation of these relationships can provide for discovery of therapeutic compositions useful in mitigating the effects of Alzheimer's Disease.